Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L

Abstract

The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe(2+) ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis.

Keywords: Benzoxazolinone detoxification; Zea mays L; cell wall polymer; extraplastic space; glucanase; glucoside carbamate pathway; peroxidase; protein shifting.

Figure 1.

Structures of compounds mentioned in the text. Known BOA / MBOA plant detoxification products and the fungal degradation product of glucoside carbamate, N-glucosylated carbamic acid. N-(2-hydroxyphenyl)malonamic acid (oHPMA) is a microbial degradation product of BOA, which requires BOA heterocycle ring opening as first step of the reaction sequence. N-(3-oxo-3H-phenoxazin-2-yl)-acetamide (AAPO) is a fungal product derived from BOA degradation via 2-aminophenol and N-(3-oxo-3H-) phenoxazin-2-one.

Figure 2.

Accumulation of glucoside carbamate in the Arabidopsis lines 35S::Bx8, 35S::Bx9 and 35S::Co-Bx8 and the Wild Type after 24 h incubation with 0.5, 1.0, and 2.0 mM (250 ml/12 plants). Means ± SD are shown, asterisks indicate significant differences compared with the wild type Col-0 (t-test, ** p ≤ 0.001; ***p ≤ 0.0001).

Figure 3.

Benzoxoxazinone / benzoxazolinone contents of the cell wall fraction after 6 and 24 h incubation with BOA in comparison to the control. Glc Carb = glucoside carbamate. MBOA content after 24 h incubation with BOA is significantly different from the contents when the incubation was started and after 6 h (means ±SD, t-test **p ≤ 0.001; ***p ≤ 0.0001).

Figure 4.

HPLC chromatograms of BOA media after 24 h incubation of maize seedlings. Above: Medium without BNAP (control), below medium with 500 µM BNAP. The inhibitor prevented the degradation/detoxification of benzoxazinones (HMBOA, DIMBOA) and benzoxazolinones (BOA, MBOA). The fungal BOA degradation product oHPMA (N-(2-hydroxylphenyl)malonamic acid) and the phenoxazinone, found in the control medium are not produced in presence of the inhibitor.

Figure 5.

Compounds detected in incubation media supplemented with 30 µM BNAP and in control media. BNAP led to an accumulation of glucoside carbamate and a reduced degradation rate of BOA. In the controls, the fungal detoxification product oHPMA, AAPO (Phen) and only 50% of the applied BOA were found.

Figure 6.

Influence of BNAP concentrations on glucoside carbamate accumulation in the root. An increase was detected with 15 and 20 µmol BNAP. 30 µmol of the effector reduced the amount of glucoside carbamate, 50 µmol led to a complete inhibition. Means ± SD are shown, asterisks indicate significant differences compared with the control (t-test, ** p ≤ 0.001; ***p ≤ 0.0001).

Figure 7.

Reduced glucoside carbamate contents in roots which were incubated with 0.5, 1.0 and 2.5 mM 2,3-butanedione added to the BOA medium. Means ± SD are shown, asterisks indicate significant differences compared with the control (t-test, ** p ≤ 0.001; ***p ≤ 0.0001). 2.5 mM 2,3-butanedione led to almost no glucoside carbamate, but since 2.5 mM started to affect benzoxazinone biosynthesis, results obtained with 2.5 mM were not further considered. Means ± SD are shown, asterisks indicate significant differences compared with the control (t-test, ***p ≤ 0.0001).

Figure 8.

Glucoside carbamate synthesis within 60 min and in overnight-assays with highly concentrated total cell protein extracts. A: whole protein fraction from roots of 24 h BOA incubated seedlings, H₂O₂, Fe²⁺. B: whole protein fraction + apoplast fraction + cellobiose, laminarin and cell wall material, H₂O₂, Fe²⁺, C: as A, but without Fe²⁺. D: as A but with 1 mM 2,3-butanedione; E: as A but without H₂O₂. A-E: assays were incubated for 1 h at 30°C.

Figure 9.

Western blots of cell wall, cytosolic and apoplast associated BX9. Fragments of BX9 are mainly located in the cytosol. Lane BX9: control, recombinant protein; M: marker proteins, protein mass in k_D. Samples were prepared after 3 and 24 h of BOA incubation, and an additional apoplastic sample after 48 h. The recombinant BX9 protein shows a major band at 42 kD and a weak degradation product at 40 kD. These bands are seen in the cytosol samples together with 2 other degradation products at 23 and 21 k_D. In the apoplast samples only the 40 k_D band is present, which also the major immune stained band in the wall samples.

Figure 10.

Carbohydrate related enzymes in the apoplastic fluid and cell wall. Gray bars: control, black bars: BOA. Genome Sequence IDs are below the bars. Figure 10: Carbohydrate related enzymes in the apoplastic fluid and cell wall. Gray bars: control, black bars: BOA; Genome Sequence IDs are below the bars (MaiZeGBD maize genetics and genomics data base).

Figure 11.

Peroxidases in the apoplastic fluid (A,B) and the salt extracted cell wall fraction (C,D). Gray bars: control, black bars: BOA. Genome Sequence IDs are below the bars. *: new, ┴: not present in BOA samples. Arrows upwards: increased abundance in BOA samples; arrows downwards: decreased abundance in BOA samples.

Figure 12.

Glutathione transferases in the apoplastic fluid, AP (A) and the salt extracted cell wall fraction, CW (B). Gray bars: control, black bars: BOA. Genome Sequence IDs are below the bars. *: new in the fraction.

Figure 13.

Influence of ethacrynic acid (E, GST inhibitor) and of the transporter inhibitors Nifedipine (N), Verapamil (V) on glucoside carbamate accumulation. Ethacrynic acid and nifedipine increase the accumulation significantly compared to the control (means ±SD; E: p < 0.0001; N: p < 0.001). C: control.

Figure 14.

Cell wall components in different zones of roots of BOA treated and untreated plants. The amount of Klason lignin (light gray) is increased in the younger root parts of BOA treated plants (left hand side). Soluble lignin (gray), cellulose (dark gray) and hemicellulose (black) do not show significant differences in the older root sections but within the first 1 cm from the root tip. The horizontal bars indicate the root sections which were used for the proteome analyses. Shown are means ±SD; *p < 0.005.

Figure 15.

BOA and BOA detoxification products in the roots of the maize mutant BX-less without and in presence of 50 µM BNAP. gentiob. carbamate = gentiobioside carbamate; tr = traces. C: controls; BNAP: incubations with BNAP. Glucoside carbamate is slightly more hydrophilic (retention time 12.2 min) than BOA-N-glucoside (retention time 15.25 min). Shown are means ± SD of 3 biological repetitions.

Figure 16.

Partly hypothetical pathway of glucoside carbamate synthesis in the extraplastic space, malonylation and import into the vacuole for temporary storage, export and transformation/reconstitution to BOA by the endophyte Fusarium verticillioides. Peroxidase generated BOA radicals and subsequent fixation of the radicals at carbohydrate cell wall components are thought to present the major start reactions for later N-glucosylated BOA release by hydrolytic enzymes. A rearrangement of the N-glucosylated molecule results in glucoside carbamate, which can be malonylated and stored within the vacuole. This pathway prevents not only entering of toxic benzoxazolinone into the cytosol, but is competitive to phenoxazinone synthesis. An auxiliary pathway may start after generation of instable carbamic acid and N-glucosylation by maize glucosyltransferase BX9. Fusarium verticillioides can open the lactone ring of glucoside carbamate, resulting in glucosylated carbamic acid. This relatively stable compound was recently identified as a fungal degradation product of glucoside carbamate. It can be further degraded resulting finally in phenoxazinones or is recycled to BOA.